The role of a novel scaffold protein in mediating RPE phagocytosis of photoreceptor outer segment Project Summary/Abstract: Vision relies on the structural and functional interplay between photoreceptors (PR) cells and the overlying retinal pigment epithelium (RPE). RPE cells, as highly-specialized phagocytes, are responsible for maintaining retinal homeostasis through mediating numerous intra- and intercellular interactions central for retinal survival and function. One of the most important functions of RPE is to actively participate in the daily phagocytosis of shed PR outer segment (OS) tips. Because of the diurnal rhythm of phagocytosis, any dysfunction in RPE cells hampering efficient photoreceptor clearance results in abnormal accumulation of phagocytic debris in the intracellular and extracellular space; a hallmark of age-related retinopathies. Consequently, a chronic delay in OS ingestion and degradation contributes to RPE cell damage, and ultimately, to the irreversible loss of vision. Understanding the complex molecular pathways along with backup mechanisms utilized by RPE will open new avenues toward development of treatment strategies aiming at restoration of RPE cells homeostasis and prevention of age-related RPE dysfunction. In these studies, we will investigate a novel regulator of the RPE phagocytic machinery, a large scaffold protein AHNAK, so far overlooked in the retina. AHNAK is a binding partner of Annexin A2, a key mediator of cytoskeletal dynamics critical for temporal regulation of OS phagocytosis. Our preliminary studies indicate that AHNAK occupies the most strategic retinal location at the RPE-PR interface, and strongly suggest that this large protein plays important functional role(s) in the RPE-mediated phagocytosis. Based on our pilot findings, we hypothesize that AHNAK is necessary for synchronized phagocytosis by aiding in coordination of OS ingestion and phagosome maturation, and predict that loss of AHNAK function results in delayed OS degradation and phagosome accumulation; hallmarks of age-related RPE dysfunction. Our long-term goal is to understand AHNAK's role in the regulation of daily OS phagocytosis, and its contribution to the maintenance of retinal health. To our knowledge, these studies are the first to address AHNAK's function in the retina, and the first to develop Ahnak-/- mouse model toward understanding RPE homeostasis. Here, we will explore the intriguing possibility that AHNAK may constitute a missing link in RPE-mediated phagocytosis, necessary for both the temporal regulation of OS phagocytosis and for MREG/LC3B-mediated phagosome maturation. We expect that deciphering the molecular and physiological function of this new scaffold protein in RPE homeostasis will provide valuable tools for identification of novel therapeutics for age-related disorders of the RPE and retina.